Cisplatin is a widely used anticancer drug but its efficacy is limited due to intrinsic and acquired cellular resistance. Recent studies indicate that copper homeostatic proteins, particularly copper transport pumps and chaperones, may mediate cisplatin resistance. The details of this relationship are not fully understood. The proposed study will examine the interaction between cisplatin and regulatory copper binding domains (MBDs) from the secretory pathway copper pump ATP7B (the Wilson disease protein) and the human copper chaperone Atox1. Preliminary data indicate that cisplatin can directly bind these proteins, all of which share a common fold and bind Cu(I) via conserved cysteine motifs. Subsequent experiments will identify conditions that produce stable and homogenous cisplatin-protein adducts, determine the stoichiometry of cisplatin binding and the nature of the platinum binding site as well as its effect on the overall structure of these proteins. Studies with a less complex ATP7B homolog, Archaeoglobus fulgidus CopA, will elucidate the functional consequences of cisplatin interaction with MBDs for copper binding, ATP hydrolysis, and ion transport. The information gained in this work will provide molecular level characterization of the interaction between cisplatin and copper transporters and could impact the development of new therapeutic approaches. PUBLIC HEALTH RELEVANCE: This project will result in molecular level characterization of the interaction between cisplatin, a widely prescribed anticancer drug, and copper transporters. The information gained will provide insight into the mechanism of drug resistance mediated by copper homeostasis machinery and could impact the development of new therapeutic approaches.